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Abstract [Objectives] This study was conducted to optimize the germplasm resources of Gastrodia elata and improve the condition of declining G. elata production.
[Methods] A scientific and reasonable method was used to screen the optimum nutritional conditions （carbon sources， nitrogen sources， microelements， vitamins） and environmental conditions （pH， temperature， light） of Armillaria mellea A9 growth， so as to screen the medium suitable for the growth of A. mellea.
[Results] Under the semisolid culture condition， the suitable pH range of A. mellea mycelia was 4.0-9.0; the temperature was about 25 ℃; and the light condition was dark culture. Under the experimental conditions， the optimal carbon source was 25 g/L ethanol; the optimal nitrogen source was 2.5 g/L soy peptone; and the optimum microelement and vitamin were 0.5 g/L MnSO4， 0.5 g/L MgSO4 and 20 mg/L vitamin B6， respectively. After screening， the growth rate of A. mellea rhizomorph was as high as 13.52 mm/d， and the dry weight of mycelium also reached 0.296 7 g.
[Conclusions] This study not only omitted a lot of tedious experiments， but also obtained reliable and scientific experimental results， and achieved the purpose of rejuvenating A. mellea， which is conducive to the further development and utilization of G. elata and its symbiotic fungi.
Key words Armillaria mellea mycelial rhizomorph; Semisolid culture; Growth rate; Dry weight
Armillaria mellea， belonging to Armillaria （Fr.∶Fr.） Staude of Tricholomataceae in Hymenomycetes of Basidiomycota， is a kind of edible and medicinal fungus， which is also one of the tonic traditional Chinese medicines for disease prevention and health care. A. melleahas has sedative[2]， blood sugar lowering[3]， antitumor[4]and antiaging effects[5]， and no toxic side effects[6]. There are a variety of A. mellea strains， but the strains suitable for stable and high yield production of Gastrodia elata are few and easy to degrade[7]. Therefore， screening the media suitable for the growth of A. mellea to achieve the purpose of rejuvenating A. mellea is a problem urgent to be solved. Studies have shown that the mycelia and fermentation broth of A. mellea have similar pharmacological effects and clinical effects compared to G. elata[8]， so whether or not to replace G. elata with A. mellea has become a concern of scholars. In this study， a new method was used to screen the semisolid culture conditions of A. mellea， in order to optimize the germplasm resources and improve the yield of G. elata. This study provides reference for further development and utilization of G. elata and its symbiotic fungi. Materials and Methods
Test material
The test fungus was A. mellea A9 strain preserved in the FungalDepartment of the Institute of Medicinal Plant Development.
Test methods
Activation of culture
The A. mellea A9 strain taken out from the refrigerator was placed at room temperature overnight， then inoculated on a clean bench in a laboratory to a conventional wheat bran semisolid medium by the plating method and cultured in a dark condition in the culture chamber at 25 ℃. After the mycelium grew all over the entire dish， it can be used for inoculation.
Culture of rhizomorph
The activated A. mellea A9 was perforated on a clean bench with a perforation diameter of 8 mm， obtaining the strain pieces for later use. Then， 10 ml of the media prepared under various indices were autoclaved for 30 min （0.12-0.15MPa， 121-126 ℃）， and poured into sterile culture dishes， respectively. The 8 mm strain pieces were inoculated in the middle of the culture dishes， respectively， followed by sealing with a sealing film.
Screening of media
Wheat bran medium
Wheat bran 50 g （filtrate， which was obtained by boiling the wheat bran for half an hour and filtering with three layers of gauze）， glucose 20 g， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g and agar 7.5 g were mixed and volumed with 1 000 ml of distilled water， obtaining the medium with a natural pH.
PDA medium
Potato 200 g （filtrate， which was obtained by boiling the potato for half an hour and filtering）， glucose 20 g and agar 7.5 g were mixed and volumed with 1 000 ml of distilled water， obtaining the medium with a natural pH.
Wheat bran+sawdust medium
On the basis of the wheat bran medium， 20 g of sawdust was added per 1 000 ml of the medium.
PDA+sawdust medium
On the basis of the PDA medium， 20 gof sawdust was added per 1 000 ml of the medium.
Screening of cultivation environments
pH value
The wheat bran medium was prepared， and the pH of the medium was adjusted to such 15 pH values as 2.0， 3.0， 4.0， 4.5， 5.0， 5.5， 6.0， 6.5， 7.0， 7.5， 8.0， 9.0， 10.0， 11.0 and 12.0 with 1.0 g/L NaOH and 1.0 mol/L H2SO4， respectively， in five repetitions for each pH treatment. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed. Temperature
The wheat bran medium was prepared， and the fungus was cultured at nine temperatures as 5， 10， 16， 19， 22， 25， 28， 31 and 34 ℃， respectively， in five repetitions for each temperature treatment. The culture was performed for 20 d in different constant temperature incubators in dark. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the mycelial rhizomorph was also observed.
Illumination
Using wheat bran medium， three treatments， i.e.， continuous light culture， continuous dark culture and 12 h/12 h alternate light and dark culture were set up， in 5 replicates. The fungus was cultured for 20 d at 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Screening of nutritional factors
Carbon source
The selected carbon sources for the medium were glucose， maltose， lactose， sucrose， soluble starch， corn flour， mannitol and ethanol， of which 15， 20 and 25 g were taken， respectively. If the amount of the carbon source was not enoughto screen for the best carbon source， it can be increased by the difference of 5.0 g. Meanwhile， the medium without carbon source was used as a control. In addition to the carbon source， soy peptone 2.0 g， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Nitrogen source
The selected nitrogen sources for the medium were soy peptone， yeast extract， beef extract， urea， casein hydrolysate， KNO3 and （NH4）2SO4， of which 1.5， 2.0 and 2.5 g were taken， respectively. If the amount of the nitrogen source was not enough to screen for the best carbon source， it can be increased by the difference of 0.5 g. Meanwhile， the medium without nitrogen source was used as a control. In addition to the nitrogen source， the best carbon source selected， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g， agar 7.5 g and distilled water 1 000ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed. Microelements
The selected microelements for the medium were CuSO4， MgSO4， ZnSO4， Fe2（SO4）3 and MnSO4， of which 0.5， 1.0 and 1.5 g were taken， respectively. If the amount of the microelement was not enough to screen for the best value， it can be increased by the differences of 0.25 and 0.50 g. Meanwhile， the medium without microelement was used as a control. In addition to the microelement， the best carbon source and nitrogen source that had been screened， potassium dihydrogen phosphate 2.0 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Vitamins
The selected vitamins for the medium were VB1， VB2， VB6， Vc and compound VB， the amounts of which were 10， 20 and 30 mg/L， respectively. If the amount of the microelement was not enough to screen for the best value， it can be increased by the difference of 10 mg/L. Meanwhile， the medium without vitamin was used as a control. In addition to vitamin， the best carbon source， nitrogen source and microelement that had been screened， potassium dihydrogen phosphate 2.0 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH value remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Method for measuring dry weight of the fungus
The A. mellea mycelum and rhizomorph cultured on a semisolid medium 25 ℃ for 20 d under constant temperature in dark condition was washed with distilled water on a 60mesh stainless steel sieve， to thoroughly wash the attached semisolid agar away. The surface moisture was absorbed by filter paper， and the rhizomorph was dried at 60 ℃ to constant weight and weighed， obtaining the weight which was the dry weight of A. mellea mycelial rhizomorph.
Results and Analysis
Medium types
It can be seen from Table 1 that the growth rate of A. mellea A9 was better on the natural media， and there were no significant differences in the growth rate of mycelium and the dry weight of mycelium. There were many rhizomorph branches on the wheat bran medium， which might be due to the crude protein and cellulose contained in the wheat bran， which can promote the germination of the A. mellea rhizomorph. After the addition of sawdust， the growth rate and growth vigor of the mycelium increased significantly， and the number of mycorrhizal branches increased slightly， which might be due to that lignin and hemicellulose in the sawdust could increase the dry weight of A. mellea rhizomorph. A. mellea mycelial rhizomorph can grow in the range of pH 3.0-11.0， but the effects of different pH on mycelial growth were slightly different. When the pH was in the range of 4.0-9.0， the mycelial rhizomorph grew faster and the growth vigor was better， so it is the optimum pH range for the growth of A. mellea mycelial rhizomorph. In this range， different pH treatments showed no significant differences in the growth rate of rhizomorph and dry weight of mycelum and rhizomorph. When the pH was 4.0， the growth rate of rhizomorph was （15.02±0.24） mm/d， and the dry weight of mycelial rhizomorph was （0.377 1±0.060 4） g. Not only the growth rate of rhizomorph was high， but also the growth vigor of mycelium was extremely strong， so the optimum pH value was 4.0.When the pH was greater than 9.0， although the rhizomorph grew still fast， the rhizomorph became thinner and thinner， and the branches were less and less， so the growth vigor was poor. When pH < 3.0 and pH > 11.0， the A. mellea rhizomorph stopped growing. In summary， the A. mellea mycelial rhizomorph was adaptive to a wide range of pH values， and can be grown at different pH values except peracid and overbase.
Temperature
It can be seen from Fig. 2 that the A. mellea mycelial rhizomorph can grow in the temperature range of 16-28 ℃， but the effects of different temperatures on the growth of rhizomorph were different. When the culture temperature was between 16 and 25 ℃， the growth rate of rhizomorph was lower， and the growth of mycelium was the most vigorous at 19 ℃; and with the increase of the culture temperature， the growth rate of rhizomorph gradually increased， the growth vigor was gradually enhanced， and the peak values were reached at 25 ℃ （the growth rate of rhizomorph was （11.87±0.10） mm/d， and the dry weight of mycelial rhizomorph was （0.197 3±0.016 0） g）. When the temperature was higher than 25 ℃， the growth rate and dry weight of A. mellea rhizomorph were on the decrease. At 28 ℃， the rhizomorph aged faster， and there was a tan scablike matter. When the culture temperature was less than 10 ℃ or greater than 28 ℃， the A. mellea mycelial rhizomorph did not grow. The A. mellea strain cultured at 10 ℃or 31 ℃ for 20 dwas recultured at 25 ℃， and the results showed that the strain that had been cultured at 10 ℃ stared to grow， while the stain that had been cultured at 31 ℃ still did not grow. It can be seen from the results of this experiment that the temperature range suitable for the growth of A. mellea is narrow， and the temperature should be controlled at about 25 ℃. Low temperature can keep A. mellea active， while high temperature will completely inactivate A. mellea. This suggests that we must strictly control the culture conditions of A. mellea in the preservation and production. Illumination
It can be seen from Fig. 3 and Table 2 that the effects of different light conditions on the growth of A. mellea differed significantly. Under the conditions of continuous illumination and 12 h/12 halternate light and dark culture， the average growth rates of rhizomorph were （5.39 ± 0.06） and （6.25 ± 0.43） mm/d， respectively， and the dry weights of mycelial rhizomorph were significantly different， at （0.084 5 ± 0.037 8） and （0.089 1 ± 0.035 4） g， respectively. However， under the continuous light condition， the rhizomorph aged severely and there were fewer branches. It can be seen that visible light can cause irritation to the growth of A. mellea， resulting in a decrease in branches. Under the continuous dark culture condition， the growth rate of rhizomorph and the dry weight of mycelial rhizomorph were significantly different from other two conditions， and were （11.87±0.10） mm/d and （0.197 3±0.016 0） g， respectively. Under this light condition， the A. mellea rhizomorph was thick and strong with many branches， not prone to aging， and had a fast growth rate and good growth vigor. In view of the average growth rate， growth vigor and dry weight of mycelial rhizomorph， continuous dark culture was the best culture condition of A. mellea， which is also consistent with the growth characteristics of most fungi.
Carbon source
It can be seen from Fig. 5 that the carbon source was a nutritive material necessary for the growth of A. mellea. When a single carbon source was added， the growth rate and growth vigor of A. mellea mycelial rhizomorph were significantly inferior to those in natural media such as wheat bran and PDA. A. mellea had strong selectivity to carbon sources， and when the disaccharides such as lactose and sucrose were used as carbon sources， the A. mellea mycelial rhizomorph did not grow. Inconsistent with literatures， the promoting effect of mannitol on the growth of A. mellea A9 mycelial rhizomorph was not ideal， and the high concentration of mannitol even inhibited the growth of rhizomorph. When glucose and maltose were used as carbon sources， the A. mellea rhizomorph was thicker， but the branches were fewer， and growth rate was lower. From Table 3， it can be seen that when using corn flour as the carbon source， the growth rate of mycelium and the dry weight of mycelium were better， but the rhizomorph was extremely fine and suffered from and serious aging phenomenon. In summary， when ethanol （25 g/L） was used as the carbon source， the aerial mycelia were developed， and the rhizomorph had more branches， grew strongly and faster with the average growth rate reaching （12.32±1.04）mm/d， which was significantly different from other treatments; and the dry weight of mycelial rhizomorph could reach （0.155 1±0.012 2） g.Therefore， ethanol was the best carbon source for the growth of A. mellea mycelial rhizomorph. Nitrogen source
It can be seen from Fig. 5 that nitrogen source was a nutritive material necessary for the growth of A. mellea， and the utilization effect of organic nitrogen by A. mellea mycelial rhizomorph was significantly better than that of the inorganic nitrogen. When KNO3 was used as the nitrogen source， the A. mellea mycelial rhizomorph did not grow. When （NH4）2SO4 was used as the nitrogen source， only the mycelia germinated， and the rhizomorph did not grow. The A. mellea grew on the culture media of the five organic nitrogen sources， and different concentrations of organic nitrogen sources had different effects on the growth of A. mellea. According to Table 4， when beef extract， casein hydrolysate， yeast extract and urea were used as nitrogen sources， the growth rate of mycelium showed no significant differences， but with urea as the nitrogen source， the A. mellea suffered from serious aging， and had a small dry weight. Combined with the figure， it can be concluded that when soy peptone was used as the nitrogen source （2.5 g/L）， the germination of A. mellea mycelium was better， the growth rate of rhizomorph and the dry weight of mycelium were （13.49±0.81） mm/d and （0.222 6±0.042 7） g， respectively， which were significantly better than other nitrogen sources， and the rhizomorph was curled， stout， and branched more， so the 2.5 g/L soy peptone was the best nitrogen source for the growth of A. mellea.
Microelement
It can be seen from Fig. 6 that the A. mellea mycelial rhizomorph can still grow under the condition of no microelements， but the rhizomorph was fine， indicating that the microelements can make the A. mellea rhizomorph grow thick. Different microelements had different effects on the growth and dry weight of A. mellea rhizomorph. When 0.5 g/L MnSO4 was added， the growth rate of rhizomorph was the fastest， up to （13.17±0.26） mm/d， but it was not significantly different from 0.5 g/L MgSO4， under which the dry weight of mycelium was （0.222 6±0.036 7） g. The second was MgSO4， which significantly promoted the elongation of mycelia， and also produced the dry weight of mycelium higher than that without the addition of microelements. The treatments with CuSO4 and ZnSO4 as microelements were the worst， and the mycelial rhizomorph did not grow. When Fe2（SO4）3 was used as an added microelement， it also had a certain inhibitory effect on the growth of A. mellea A9 mycelial rhizomorph. Therefore， within the range of microelements selected in this experiment， 0.5 g/L MnSO4 and 0.5 g/L MgSO4 were the most suitable microelements for the growth of A. mellea mycelium. Vitamin
A. mellea can grow in semisolid media added with the five kinds of test vitamins and without vitamins， but different vitamins differed in the promoting effect on the growth of mycelial rhizomorph. It can be seen from Fig. 7 and Table 6 that when 20 mg/L vitamin B6 was added， the strain grew strongly with many branches， and the growth rate was the highest， reaching （13.52±0.41） mm/d， which was significantly different from other treatments.
Meanwhile， the dry weight of mycelium was （0.296 7±0.028 0） g. The A. mellea mycelial rhizomorph could grow in three concentrations of compound vitamin B medium， but was slightly inhibited， and the growth vigor was not as good as the medium without vitamin added. It can also be seen from Table 6 that when vitamins were added at a concentration of 20 mg/L， the five vitamins all exhibited the fastest growth rates and the maximum dry weights， so 20 mg/L was a suitable concentration of vitamins for A. mellea. Therefore， 20 mg/L of vitamin B6 was the best vitamin for the growth of A. mellea in the range of vitamins selected for this study.
Conclusions and Discussion
The innovation of this study is adopting a more scientific and reasonable method to screen the nutritive factors of different concentrations of carbon sources， nitrogen sources， microelements and vitamins one by one， which can obtain reliable and scientific experimental results while saving a large number of tedious and cumbersome orthogonal experiments. Meanwhile， the wheat bran medium was also used to screen out the most suitable environmental factors such as temperature， pH value and illumination for the growth of A. mellea mycelial rhizomorph. The results of this study showed that under the semisolid culture condition， the suitable pH range of A. mellea mycelia was 4.0-9.0， and the optimal pH value was 4.0; the A. mellea mycelia had higher requirements on temperature condition， and the best temperature was about 25 ℃; and like most fungi， A. mellea mycelia needed to be protected from light. Under the experimental conditions， the best carbon source was 25 g/Lethanol， while the effect of mannitol was not good， which is slightly different from the experimental results of Cheng[9]， which might be because that different types of A. mellea differ in the demand to carbon source. The optimum nitrogen source was 2.5 g/Lsoy peptone. The optimum microelements and vitamin were 0.5 g/L MnSO4， 0.5 g/L MgSO4 and 20 mg/L vitamin B6， respectively. Under the optimal culture conditions， the growth rate of A. mellea was （13.52±0.41） mm/d; the aerial mycelia were developed with strong rhizomorph and more branches; and the dry weight of mycelium can reach （0.296 7±0.028 0） g. The overall growth status was better than that on natural synthetic media such as wheat bran and PDA alone. However， after adding sawdust to wheat bran and PDA， the growth rate and dry weight of A. mellea were significantly improved， and the number of mycorrhizal branches increased slightly as well， which might be because that lignin and hemicellulose in sawdust can promote the growth of A. mellea mycelia， which is consistent with the results of Shi et al.[10]. References
[1] ZANG JP， YUAN S， LIAN B. Research progress on Armillaria mellea （Vahl） P. Kumm[J]. Studies of Trace Elements and Health， 2004， 21（3）： 47-50. （in Chinese）
[2] ZOU R， KANG JC. Research progress on Armillaria mellea （Vahl） P. Kumm[J]. Journal of Mountain Agriculture and Biology， 2005， 24（3）： 260-264. （in Chinese）
[3] XU BK， ZHANG Y. The prevention and treatment of polysaccharide from the rhizomorph of Armillaria mellea on diabetic cataract in rat[J]. J Agric Sci Technol， 2014， 15（7）： 1086-1088.
[4] WU J， ZHOU J， LANG Y， et al. A polysaccharide from Armillaria mellea exhibition strong in vitro anticancer activity via apoptosisinvolved mechanisms[J]. Int J Med Mushrooms， 2012， 51（4）： 663-667.
[5] CHEN LY， ZHANG YX， YU M， et al. Antiaging mechanism of polysaccharide from rhizomorph of Armillaria mellea in Caenorhabditis elegans[J]. Chinese Traditional and Herbal Drugs， 2013， 44（4）： 449-453. （in Chinese）
[6] YU M， SHEN YS. The effect of polysaccharide from the rhizomorph of Armillaria mellea （AMP） on the blood glucose and acutely toxic in mice[J]. Edible Fungi of China， 2002， 21（1）： 35-37. （in Chinese）
[7] WANG QY， GUO SX， GUAN FB. Studies on influences of Armillaria mellea strains from different sources on yield of Gastrodia elata[J]. Chinese Traditional and Herbal Drugs，2001，32（9）： 839-841. （in Chinese）
[8] TAN ZJ， XIE DP， WANG Z， et al. Studies on the isolation， purification and properties of polysaccharide from Armillaria mellea[J]. Food Science， 2002， 23（9）： 49-52. （in Chinese）
[9] CHENG XH， GUO SX. Cultural characteristics of Armillaria mellea on solid media[J]. Acta Academiae Medicinae Sinicae， 2006， 28（04）： 553-557. （in Chinese）
[10] SHI HY， CUI W， ZHENG HC， et al. Research on biological characteristics of Armillaria mellea[J]. Journal of Fungal Research， 2014， 12（4）： 229-232. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] A scientific and reasonable method was used to screen the optimum nutritional conditions （carbon sources， nitrogen sources， microelements， vitamins） and environmental conditions （pH， temperature， light） of Armillaria mellea A9 growth， so as to screen the medium suitable for the growth of A. mellea.
[Results] Under the semisolid culture condition， the suitable pH range of A. mellea mycelia was 4.0-9.0; the temperature was about 25 ℃; and the light condition was dark culture. Under the experimental conditions， the optimal carbon source was 25 g/L ethanol; the optimal nitrogen source was 2.5 g/L soy peptone; and the optimum microelement and vitamin were 0.5 g/L MnSO4， 0.5 g/L MgSO4 and 20 mg/L vitamin B6， respectively. After screening， the growth rate of A. mellea rhizomorph was as high as 13.52 mm/d， and the dry weight of mycelium also reached 0.296 7 g.
[Conclusions] This study not only omitted a lot of tedious experiments， but also obtained reliable and scientific experimental results， and achieved the purpose of rejuvenating A. mellea， which is conducive to the further development and utilization of G. elata and its symbiotic fungi.
Key words Armillaria mellea mycelial rhizomorph; Semisolid culture; Growth rate; Dry weight
Armillaria mellea， belonging to Armillaria （Fr.∶Fr.） Staude of Tricholomataceae in Hymenomycetes of Basidiomycota， is a kind of edible and medicinal fungus， which is also one of the tonic traditional Chinese medicines for disease prevention and health care. A. melleahas has sedative[2]， blood sugar lowering[3]， antitumor[4]and antiaging effects[5]， and no toxic side effects[6]. There are a variety of A. mellea strains， but the strains suitable for stable and high yield production of Gastrodia elata are few and easy to degrade[7]. Therefore， screening the media suitable for the growth of A. mellea to achieve the purpose of rejuvenating A. mellea is a problem urgent to be solved. Studies have shown that the mycelia and fermentation broth of A. mellea have similar pharmacological effects and clinical effects compared to G. elata[8]， so whether or not to replace G. elata with A. mellea has become a concern of scholars. In this study， a new method was used to screen the semisolid culture conditions of A. mellea， in order to optimize the germplasm resources and improve the yield of G. elata. This study provides reference for further development and utilization of G. elata and its symbiotic fungi. Materials and Methods
Test material
The test fungus was A. mellea A9 strain preserved in the FungalDepartment of the Institute of Medicinal Plant Development.
Test methods
Activation of culture
The A. mellea A9 strain taken out from the refrigerator was placed at room temperature overnight， then inoculated on a clean bench in a laboratory to a conventional wheat bran semisolid medium by the plating method and cultured in a dark condition in the culture chamber at 25 ℃. After the mycelium grew all over the entire dish， it can be used for inoculation.
Culture of rhizomorph
The activated A. mellea A9 was perforated on a clean bench with a perforation diameter of 8 mm， obtaining the strain pieces for later use. Then， 10 ml of the media prepared under various indices were autoclaved for 30 min （0.12-0.15MPa， 121-126 ℃）， and poured into sterile culture dishes， respectively. The 8 mm strain pieces were inoculated in the middle of the culture dishes， respectively， followed by sealing with a sealing film.
Screening of media
Wheat bran medium
Wheat bran 50 g （filtrate， which was obtained by boiling the wheat bran for half an hour and filtering with three layers of gauze）， glucose 20 g， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g and agar 7.5 g were mixed and volumed with 1 000 ml of distilled water， obtaining the medium with a natural pH.
PDA medium
Potato 200 g （filtrate， which was obtained by boiling the potato for half an hour and filtering）， glucose 20 g and agar 7.5 g were mixed and volumed with 1 000 ml of distilled water， obtaining the medium with a natural pH.
Wheat bran+sawdust medium
On the basis of the wheat bran medium， 20 g of sawdust was added per 1 000 ml of the medium.
PDA+sawdust medium
On the basis of the PDA medium， 20 gof sawdust was added per 1 000 ml of the medium.
Screening of cultivation environments
pH value
The wheat bran medium was prepared， and the pH of the medium was adjusted to such 15 pH values as 2.0， 3.0， 4.0， 4.5， 5.0， 5.5， 6.0， 6.5， 7.0， 7.5， 8.0， 9.0， 10.0， 11.0 and 12.0 with 1.0 g/L NaOH and 1.0 mol/L H2SO4， respectively， in five repetitions for each pH treatment. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed. Temperature
The wheat bran medium was prepared， and the fungus was cultured at nine temperatures as 5， 10， 16， 19， 22， 25， 28， 31 and 34 ℃， respectively， in five repetitions for each temperature treatment. The culture was performed for 20 d in different constant temperature incubators in dark. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the mycelial rhizomorph was also observed.
Illumination
Using wheat bran medium， three treatments， i.e.， continuous light culture， continuous dark culture and 12 h/12 h alternate light and dark culture were set up， in 5 replicates. The fungus was cultured for 20 d at 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Screening of nutritional factors
Carbon source
The selected carbon sources for the medium were glucose， maltose， lactose， sucrose， soluble starch， corn flour， mannitol and ethanol， of which 15， 20 and 25 g were taken， respectively. If the amount of the carbon source was not enoughto screen for the best carbon source， it can be increased by the difference of 5.0 g. Meanwhile， the medium without carbon source was used as a control. In addition to the carbon source， soy peptone 2.0 g， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Nitrogen source
The selected nitrogen sources for the medium were soy peptone， yeast extract， beef extract， urea， casein hydrolysate， KNO3 and （NH4）2SO4， of which 1.5， 2.0 and 2.5 g were taken， respectively. If the amount of the nitrogen source was not enough to screen for the best carbon source， it can be increased by the difference of 0.5 g. Meanwhile， the medium without nitrogen source was used as a control. In addition to the nitrogen source， the best carbon source selected， potassium dihydrogen phosphate 2.0 g， magnesium sulfate 1.5 g， agar 7.5 g and distilled water 1 000ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed. Microelements
The selected microelements for the medium were CuSO4， MgSO4， ZnSO4， Fe2（SO4）3 and MnSO4， of which 0.5， 1.0 and 1.5 g were taken， respectively. If the amount of the microelement was not enough to screen for the best value， it can be increased by the differences of 0.25 and 0.50 g. Meanwhile， the medium without microelement was used as a control. In addition to the microelement， the best carbon source and nitrogen source that had been screened， potassium dihydrogen phosphate 2.0 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH remained natural. The fungus was cultured for 20 d under the constant temperature of 25℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Vitamins
The selected vitamins for the medium were VB1， VB2， VB6， Vc and compound VB， the amounts of which were 10， 20 and 30 mg/L， respectively. If the amount of the microelement was not enough to screen for the best value， it can be increased by the difference of 10 mg/L. Meanwhile， the medium without vitamin was used as a control. In addition to vitamin， the best carbon source， nitrogen source and microelement that had been screened， potassium dihydrogen phosphate 2.0 g， agar 7.5 g and distilled water 1 000 ml were added， and the pH value remained natural. The fungus was cultured for 20 d under the constant temperature of 25 ℃. The growth rate of the rhizomorph was measured with a digital vernier caliper， and the average value of the five replicates was calculated. The growth state of the rhizomorph and mycelium was also observed.
Method for measuring dry weight of the fungus
The A. mellea mycelum and rhizomorph cultured on a semisolid medium 25 ℃ for 20 d under constant temperature in dark condition was washed with distilled water on a 60mesh stainless steel sieve， to thoroughly wash the attached semisolid agar away. The surface moisture was absorbed by filter paper， and the rhizomorph was dried at 60 ℃ to constant weight and weighed， obtaining the weight which was the dry weight of A. mellea mycelial rhizomorph.
Results and Analysis
Medium types
It can be seen from Table 1 that the growth rate of A. mellea A9 was better on the natural media， and there were no significant differences in the growth rate of mycelium and the dry weight of mycelium. There were many rhizomorph branches on the wheat bran medium， which might be due to the crude protein and cellulose contained in the wheat bran， which can promote the germination of the A. mellea rhizomorph. After the addition of sawdust， the growth rate and growth vigor of the mycelium increased significantly， and the number of mycorrhizal branches increased slightly， which might be due to that lignin and hemicellulose in the sawdust could increase the dry weight of A. mellea rhizomorph. A. mellea mycelial rhizomorph can grow in the range of pH 3.0-11.0， but the effects of different pH on mycelial growth were slightly different. When the pH was in the range of 4.0-9.0， the mycelial rhizomorph grew faster and the growth vigor was better， so it is the optimum pH range for the growth of A. mellea mycelial rhizomorph. In this range， different pH treatments showed no significant differences in the growth rate of rhizomorph and dry weight of mycelum and rhizomorph. When the pH was 4.0， the growth rate of rhizomorph was （15.02±0.24） mm/d， and the dry weight of mycelial rhizomorph was （0.377 1±0.060 4） g. Not only the growth rate of rhizomorph was high， but also the growth vigor of mycelium was extremely strong， so the optimum pH value was 4.0.When the pH was greater than 9.0， although the rhizomorph grew still fast， the rhizomorph became thinner and thinner， and the branches were less and less， so the growth vigor was poor. When pH < 3.0 and pH > 11.0， the A. mellea rhizomorph stopped growing. In summary， the A. mellea mycelial rhizomorph was adaptive to a wide range of pH values， and can be grown at different pH values except peracid and overbase.
Temperature
It can be seen from Fig. 2 that the A. mellea mycelial rhizomorph can grow in the temperature range of 16-28 ℃， but the effects of different temperatures on the growth of rhizomorph were different. When the culture temperature was between 16 and 25 ℃， the growth rate of rhizomorph was lower， and the growth of mycelium was the most vigorous at 19 ℃; and with the increase of the culture temperature， the growth rate of rhizomorph gradually increased， the growth vigor was gradually enhanced， and the peak values were reached at 25 ℃ （the growth rate of rhizomorph was （11.87±0.10） mm/d， and the dry weight of mycelial rhizomorph was （0.197 3±0.016 0） g）. When the temperature was higher than 25 ℃， the growth rate and dry weight of A. mellea rhizomorph were on the decrease. At 28 ℃， the rhizomorph aged faster， and there was a tan scablike matter. When the culture temperature was less than 10 ℃ or greater than 28 ℃， the A. mellea mycelial rhizomorph did not grow. The A. mellea strain cultured at 10 ℃or 31 ℃ for 20 dwas recultured at 25 ℃， and the results showed that the strain that had been cultured at 10 ℃ stared to grow， while the stain that had been cultured at 31 ℃ still did not grow. It can be seen from the results of this experiment that the temperature range suitable for the growth of A. mellea is narrow， and the temperature should be controlled at about 25 ℃. Low temperature can keep A. mellea active， while high temperature will completely inactivate A. mellea. This suggests that we must strictly control the culture conditions of A. mellea in the preservation and production. Illumination
It can be seen from Fig. 3 and Table 2 that the effects of different light conditions on the growth of A. mellea differed significantly. Under the conditions of continuous illumination and 12 h/12 halternate light and dark culture， the average growth rates of rhizomorph were （5.39 ± 0.06） and （6.25 ± 0.43） mm/d， respectively， and the dry weights of mycelial rhizomorph were significantly different， at （0.084 5 ± 0.037 8） and （0.089 1 ± 0.035 4） g， respectively. However， under the continuous light condition， the rhizomorph aged severely and there were fewer branches. It can be seen that visible light can cause irritation to the growth of A. mellea， resulting in a decrease in branches. Under the continuous dark culture condition， the growth rate of rhizomorph and the dry weight of mycelial rhizomorph were significantly different from other two conditions， and were （11.87±0.10） mm/d and （0.197 3±0.016 0） g， respectively. Under this light condition， the A. mellea rhizomorph was thick and strong with many branches， not prone to aging， and had a fast growth rate and good growth vigor. In view of the average growth rate， growth vigor and dry weight of mycelial rhizomorph， continuous dark culture was the best culture condition of A. mellea， which is also consistent with the growth characteristics of most fungi.
Carbon source
It can be seen from Fig. 5 that the carbon source was a nutritive material necessary for the growth of A. mellea. When a single carbon source was added， the growth rate and growth vigor of A. mellea mycelial rhizomorph were significantly inferior to those in natural media such as wheat bran and PDA. A. mellea had strong selectivity to carbon sources， and when the disaccharides such as lactose and sucrose were used as carbon sources， the A. mellea mycelial rhizomorph did not grow. Inconsistent with literatures， the promoting effect of mannitol on the growth of A. mellea A9 mycelial rhizomorph was not ideal， and the high concentration of mannitol even inhibited the growth of rhizomorph. When glucose and maltose were used as carbon sources， the A. mellea rhizomorph was thicker， but the branches were fewer， and growth rate was lower. From Table 3， it can be seen that when using corn flour as the carbon source， the growth rate of mycelium and the dry weight of mycelium were better， but the rhizomorph was extremely fine and suffered from and serious aging phenomenon. In summary， when ethanol （25 g/L） was used as the carbon source， the aerial mycelia were developed， and the rhizomorph had more branches， grew strongly and faster with the average growth rate reaching （12.32±1.04）mm/d， which was significantly different from other treatments; and the dry weight of mycelial rhizomorph could reach （0.155 1±0.012 2） g.Therefore， ethanol was the best carbon source for the growth of A. mellea mycelial rhizomorph. Nitrogen source
It can be seen from Fig. 5 that nitrogen source was a nutritive material necessary for the growth of A. mellea， and the utilization effect of organic nitrogen by A. mellea mycelial rhizomorph was significantly better than that of the inorganic nitrogen. When KNO3 was used as the nitrogen source， the A. mellea mycelial rhizomorph did not grow. When （NH4）2SO4 was used as the nitrogen source， only the mycelia germinated， and the rhizomorph did not grow. The A. mellea grew on the culture media of the five organic nitrogen sources， and different concentrations of organic nitrogen sources had different effects on the growth of A. mellea. According to Table 4， when beef extract， casein hydrolysate， yeast extract and urea were used as nitrogen sources， the growth rate of mycelium showed no significant differences， but with urea as the nitrogen source， the A. mellea suffered from serious aging， and had a small dry weight. Combined with the figure， it can be concluded that when soy peptone was used as the nitrogen source （2.5 g/L）， the germination of A. mellea mycelium was better， the growth rate of rhizomorph and the dry weight of mycelium were （13.49±0.81） mm/d and （0.222 6±0.042 7） g， respectively， which were significantly better than other nitrogen sources， and the rhizomorph was curled， stout， and branched more， so the 2.5 g/L soy peptone was the best nitrogen source for the growth of A. mellea.
Microelement
It can be seen from Fig. 6 that the A. mellea mycelial rhizomorph can still grow under the condition of no microelements， but the rhizomorph was fine， indicating that the microelements can make the A. mellea rhizomorph grow thick. Different microelements had different effects on the growth and dry weight of A. mellea rhizomorph. When 0.5 g/L MnSO4 was added， the growth rate of rhizomorph was the fastest， up to （13.17±0.26） mm/d， but it was not significantly different from 0.5 g/L MgSO4， under which the dry weight of mycelium was （0.222 6±0.036 7） g. The second was MgSO4， which significantly promoted the elongation of mycelia， and also produced the dry weight of mycelium higher than that without the addition of microelements. The treatments with CuSO4 and ZnSO4 as microelements were the worst， and the mycelial rhizomorph did not grow. When Fe2（SO4）3 was used as an added microelement， it also had a certain inhibitory effect on the growth of A. mellea A9 mycelial rhizomorph. Therefore， within the range of microelements selected in this experiment， 0.5 g/L MnSO4 and 0.5 g/L MgSO4 were the most suitable microelements for the growth of A. mellea mycelium. Vitamin
A. mellea can grow in semisolid media added with the five kinds of test vitamins and without vitamins， but different vitamins differed in the promoting effect on the growth of mycelial rhizomorph. It can be seen from Fig. 7 and Table 6 that when 20 mg/L vitamin B6 was added， the strain grew strongly with many branches， and the growth rate was the highest， reaching （13.52±0.41） mm/d， which was significantly different from other treatments.
Meanwhile， the dry weight of mycelium was （0.296 7±0.028 0） g. The A. mellea mycelial rhizomorph could grow in three concentrations of compound vitamin B medium， but was slightly inhibited， and the growth vigor was not as good as the medium without vitamin added. It can also be seen from Table 6 that when vitamins were added at a concentration of 20 mg/L， the five vitamins all exhibited the fastest growth rates and the maximum dry weights， so 20 mg/L was a suitable concentration of vitamins for A. mellea. Therefore， 20 mg/L of vitamin B6 was the best vitamin for the growth of A. mellea in the range of vitamins selected for this study.
Conclusions and Discussion
The innovation of this study is adopting a more scientific and reasonable method to screen the nutritive factors of different concentrations of carbon sources， nitrogen sources， microelements and vitamins one by one， which can obtain reliable and scientific experimental results while saving a large number of tedious and cumbersome orthogonal experiments. Meanwhile， the wheat bran medium was also used to screen out the most suitable environmental factors such as temperature， pH value and illumination for the growth of A. mellea mycelial rhizomorph. The results of this study showed that under the semisolid culture condition， the suitable pH range of A. mellea mycelia was 4.0-9.0， and the optimal pH value was 4.0; the A. mellea mycelia had higher requirements on temperature condition， and the best temperature was about 25 ℃; and like most fungi， A. mellea mycelia needed to be protected from light. Under the experimental conditions， the best carbon source was 25 g/Lethanol， while the effect of mannitol was not good， which is slightly different from the experimental results of Cheng[9]， which might be because that different types of A. mellea differ in the demand to carbon source. The optimum nitrogen source was 2.5 g/Lsoy peptone. The optimum microelements and vitamin were 0.5 g/L MnSO4， 0.5 g/L MgSO4 and 20 mg/L vitamin B6， respectively. Under the optimal culture conditions， the growth rate of A. mellea was （13.52±0.41） mm/d; the aerial mycelia were developed with strong rhizomorph and more branches; and the dry weight of mycelium can reach （0.296 7±0.028 0） g. The overall growth status was better than that on natural synthetic media such as wheat bran and PDA alone. However， after adding sawdust to wheat bran and PDA， the growth rate and dry weight of A. mellea were significantly improved， and the number of mycorrhizal branches increased slightly as well， which might be because that lignin and hemicellulose in sawdust can promote the growth of A. mellea mycelia， which is consistent with the results of Shi et al.[10]. References
[1] ZANG JP， YUAN S， LIAN B. Research progress on Armillaria mellea （Vahl） P. Kumm[J]. Studies of Trace Elements and Health， 2004， 21（3）： 47-50. （in Chinese）
[2] ZOU R， KANG JC. Research progress on Armillaria mellea （Vahl） P. Kumm[J]. Journal of Mountain Agriculture and Biology， 2005， 24（3）： 260-264. （in Chinese）
[3] XU BK， ZHANG Y. The prevention and treatment of polysaccharide from the rhizomorph of Armillaria mellea on diabetic cataract in rat[J]. J Agric Sci Technol， 2014， 15（7）： 1086-1088.
[4] WU J， ZHOU J， LANG Y， et al. A polysaccharide from Armillaria mellea exhibition strong in vitro anticancer activity via apoptosisinvolved mechanisms[J]. Int J Med Mushrooms， 2012， 51（4）： 663-667.
[5] CHEN LY， ZHANG YX， YU M， et al. Antiaging mechanism of polysaccharide from rhizomorph of Armillaria mellea in Caenorhabditis elegans[J]. Chinese Traditional and Herbal Drugs， 2013， 44（4）： 449-453. （in Chinese）
[6] YU M， SHEN YS. The effect of polysaccharide from the rhizomorph of Armillaria mellea （AMP） on the blood glucose and acutely toxic in mice[J]. Edible Fungi of China， 2002， 21（1）： 35-37. （in Chinese）
[7] WANG QY， GUO SX， GUAN FB. Studies on influences of Armillaria mellea strains from different sources on yield of Gastrodia elata[J]. Chinese Traditional and Herbal Drugs，2001，32（9）： 839-841. （in Chinese）
[8] TAN ZJ， XIE DP， WANG Z， et al. Studies on the isolation， purification and properties of polysaccharide from Armillaria mellea[J]. Food Science， 2002， 23（9）： 49-52. （in Chinese）
[9] CHENG XH， GUO SX. Cultural characteristics of Armillaria mellea on solid media[J]. Acta Academiae Medicinae Sinicae， 2006， 28（04）： 553-557. （in Chinese）
[10] SHI HY， CUI W， ZHENG HC， et al. Research on biological characteristics of Armillaria mellea[J]. Journal of Fungal Research， 2014， 12（4）： 229-232. （in Chinese）
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